Lysine 77 is a Key Residue in Aggregation of Equinatoxin II, a Pore-forming Toxin from Sea Anemone Actinia equina |
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Authors: | G Anderluh A Barlič C Potrich P Maček G Menestrina |
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Institution: | (1) Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia, YU;(2) CNR-ITC Centro di Fisica degli Stati Aggregati, Via Sommarive 18, 38050 Povo (Trento), Italy, IT |
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Abstract: | Among eighteen point mutants of equinatoxin II produced in E. coli, containing a single cystein substitution at variable position, EqtIIK77C was chosen for its peculiar properties. It was almost
100 times less hemolytic than the wild-type, but its hemolytic activity could be restored by chemical modification of the
thiol group, provided that a positive charge was reintroduced. This indicates that a positive charge at this position is necessary
for toxin activity. The mutant formed larger pores as compared to the wild type, but displayed the same cation selectivity.
The pores reverted to normal size upon reintroduction of the positive charge. The conformation of EqtIIK77C and its binding
to lipid membranes, either vesicles or red blood cells, was almost normal. However the kinetics of calcein release from lipid
vesicles was substantially slower than that of the wild-type. Taken together with the different size of the pore formed, this
is an indication that mutation of Lys77 → Cys influences the normal development of the aggregate which is required for assembling
the functional pore.
Received: 18 May 1999/Revised: 18 September 1999 |
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Keywords: | : Pore-forming toxin — Sea anemone — Secondary structure — Chemical modification — Sulfhydryl reagents — Pore size — Ion selectivity — Actinia equina |
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